PROJECT SUMMARY How can the second messenger Ca2+ regulate a plethora of signaling processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion? Compartmentalized Calcium signaling is a fundamental signaling principle by which Ca2+ ions exert their stimulatory function locally in a precisely controlled spatial and temporal manner. Changes in local Ca2+ concentration within the cell are regulated through Ca2+ entry across the plasma membrane to generate ?signaling hot spots? or by releasing Ca2+ from intracellular stores such as endoplasmic reticulum (ER), mitochondria or endosomes. Changes in Ca2+ concentration are ?sensed? by Ca2+ binding proteins which relay the information into a signaling output. The primary cilium is a microtubule based organelle extending from the apical plasma membrane and shaped like an antenna. Primary cilia are enriched in a specific subset of calcium permeating ion channels called polycystins (PC1 and PC2). While the Ca2+ signaling field has made seminal progress in understanding the molecular principles of compartmentalized Ca2+ signaling in organelles such as ER and endosomes, we are still lacking a functional understanding of the primary cilium as a Ca2+ signaling organelle. Mutations in polycystin ion channels result in a variety of human diseases, ranging from congenital heart disease and laterality defects to cyst formation in multiple organs (liver, kidney and pancreas). Ca2+ is likely to function as a critical second messenger within primary cilia in all of these organs, but the functional consequences of ciliary calcium signaling remain mysterious and so do the mechanisms through which ciliary ion channels are regulated. The central goal of this project is to understand how the cilia ion channels PC1 and PC2 regulate ciliary Ca2+ levels and to determine the cell biological function of compartmentalized ciliary Ca2+ signaling. There are three specific aims. The first aim is to determine how PC1/PC2 channel activity affects ciliary Ca2+ concentration. The second aim tests the hypothesis that changes in ciliary calcium concentration regulate the permeability for proteins of the transition zone, a diffusion barrier at the base of the primary cilium. The third aim determines how the N-terminus of PC1, a 3000 amino acid long fragment decorated with multiple cell adhesion domains, regulates PC1/PC2 ion channel activity. The applicants' preliminary observations include novel unpublished methods to r?ecord PC1/PC2 channel activity and to dynamically regulate ciliary Ca2+ concentration. Completion of this project will be a critical first step in understanding the cell biological function ciliary calcium signaling. Our long term goal is to understand how dysregulation of ciliary Ca2+ dynamics cause human ciliopathies.